Knowledge of a tumor's metastatic potential is important in developing cancer treatments which maximize patient survival and quality of life. The most aggressive treatment regimes should be reserved for those patients at highest risk for rapid tumor growth and metastasis. Therefore, much effort has focused on identifying markers characteristic of the metastatic potential of carcinomas.
One type of marker for the metastatic stage of a cancer is the presence of an oncogene allele. For example, a gene associated with over 30% of mammary carcinoma and ovarian carcinoma has been identified as the neu/HER2/c-erbB2 proto-oncogene, The degree of amplification of the proto-oncogene and the overexpression of its protein product were found to correlate with the severity of disease and poor prognosis. Slamon et al., Science 244:707-712 (1989). In addition, aberrant alleles of the ras and myc gene families have been shown to be involved in the progression of human cancers, and some researchers have speculated they would be useful as prognostic indicators. Field, J. K. et al., Anticancer Res. 10: 1-22 (1990). Moreover, transfection of H-ras family oncogenes and mutant p53 alleles into recipient cells will induce metastatic potential. Liotta, L. A. et al., Cell 64: 327-36 (1991).
However, a major problem with the use of these oncogene markers is that these factors are not predictive for all types of tumors. Instead, these markers are specific to the tissue or type of tumor in which they have arisen, and will likely not have broad applicability for all types of metastatic carcinomas.
A growing body of evidence indicates that angiogenesis is essential to the progression of cancer. Angiogenesis is the sprouting of new capillaries from preexisting blood vessels. Normally, angiogenesis in mammals is confined to the reproductive system, embryogenesis and development, and repair after injury. However, angiogenesis can also occur in pathological conditions such as cancer, retinal neovascularization, neovascularization in atherosclerotic plaques, hemangiomas, arthritis, and psoriasis. See Folkman, J. New England J. of Med. 333:1757-63 (1995).
Without vascularization, tumors may remain for years as small (less than a few millimeters) asymptomatic lesions. Weidner et al. New England J. of Med. 324:1-8 (1991). Tumors which become vascularized receive increased oxygen and nutrients through perfusion. Thus, tumors which are vascularized can grow and proliferate. A tumor must constantly stimulate the growth of new capillary blood vessels in order for it to continue to grow. Additionally, angiogenesis allows the tumor cells access to the host animal's circulatory system. The new blood vessels provide a gateway for tumor cells to enter the circulation and metastasize to distant sites. Folkman, J. Natl. Cancer Inst. 82:4-6 (1990); Klagsbrunn and Soker, Current Biology 3:699-702 (1993); Folkman, J., J. Natl., Cancer Inst. 82:4-6 (1991); Weidner et al., New Engl. J. Med. 324:1-5 (1991).
In fact, the extent of neovascularity is strongly correlated with metastases in primary breast carcinoma, bladder cancer, prostrate cancer, non-small cell lung cancer, cutaneous melanomas, and uterine cervix carcinoma. Reviewed in Ferrara, N., Breast Cancer Research and Treatment 36: 127-137 (1995). In these studies, tumor specimens were histologically analyzed and the number of microvesicles manually counted. The extent of tumor mass vascularization was found to be an independent predictor of the metastatic potential, and more reliable than other prognostic markers.
These results have led researchers to speculate that tumor vascularization could be used as a diagnostic tool to predict metastasis. However, counting of microvesicles in tumor specimens, besides being labor-intensive, is a qualitative art. The method requires considerable technical training in order to obtain reliable and reproducible results. Some groups have reported difficulties in reproducing the method. Wiedner, N., Amer. J. Path. 147: 9-19 (1995). Additionally, the process of preparing specimens for histology and counting vesicles is time consuming. Therefore, the application of this technique has been limited generally to research purposes.
There thus remains a need for a rapid and objective technique that could be used generally in the clinic to assess tumor vascularization and thus predict the metastatic potential of a tumor.
Several investigators have speculated that one may be able to measure angiogenic activity in patients by quantitating the presence of angiogenic proteins. There are twelve known angiogenic proteins whose presence could potentially indicate angiogenesis. Folkman J. New England J. of Med. 333:1757-63 (1995). Of these factors, those most commonly found associated with tumors are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin growth factor-2, platelet derived growth factor, and colony stimulating factors. Other factors which are candidates for angiogenic and metastatic markers are urokinase-type plasminogen activator and plasminogen activator inhibitor-1, as well as a variety of collagenases and urokinases. Wiedner, N., Amer. J. Path. 147: 9-19 (1995).
However, to choose a generally applicable marker for angiogenesis and metastasis, it is desirable that the key factor in this process be identified. At present, the factor or factors causing tumor angiogenesis have yet to be determined. Id. It has been speculated that bFGF and VEGF act synergistically. Id. Levels of bFGF were elevated in the urine of approximately 37% of a wide range of cancer patients. Similarly, bFGF was also elevated in the serum of 10% of these patients. Highest levels of bFGF were found in patients with metastatic disease. Reviewed in Folkman J. New England J. of Med. 333:1757-63 (1995). bFGF was also abnormally high in the cerebrospinal fluid of children with brain tumors, and these high levels correlated with density of microvessels in the tumor specimens. Id.
On the other hand, different investigators have found that transfection of VEGF expression plasmids into melanoma cell lines increases their capacity for growth, angiogenesis, and metastasis when injected into mice. Claffey, K. et al., Cancer Res. 56: 172-81 (1996). A correlation of the semi-quantitative level of immunoreactive VEGF protein in tumor specimens with the extent of microvesicular development and metastatic potential has been shown in colon carcinoma patients. Takahashi, Y. et al., Cancer Res. 55: 3964-68 (1995). This study only subjectively assessed the level of VEGF staining in histochemical sections. Further, certain inconsistencies in their data led these experimenters to hypothesize that VEGF may not always be the factor responsible for angiogenesis. In a study by another group, experimental xenografts of human tumor cells in mice indicated that higher levels of vascularization and VEGF RNA were found in those xenografted tumors derived from patients with a lower survival rate. Berger, D. P. et al., Annals. of Oncology 6: 817-825 (1995). However, this study did not contain a negative control, and was a retrospective analysis of tumor cells which had been serially passaged in nude mice up to 8 to 12 times, thus raising the possibility that tumor characteristics had changed.
Finally, some investigators assert that no one mechanism or factor is responsible for the development of tumor angiogenesis. While tumors might release angiogenic molecules, they assert that it is unclear how this release of factors differs from non-angiogenic cells. Instead, these investigators believe that the onset of angiogenic activity is determined by the balance of factors which are present. Folkman, J. and Shing, Y. J. Biol. Chem. 267: 10931-34 (1992).
Thus, a controversy exists over which are the best markers for tumor angiogenesis and metastatic potential. At best, there is a debate over the marker or markers which will be universally applicable to all types of solid tissue carcinomas. The present invention provides a resolution for this debate, completely eliminates the need for laborious histological analysis of tumor specimens, and provides a generally applicable and rapid method for determining the metastatic potential of solid tissue carcinoma in a patient.